Abstract
Here we describe the protocols to characterize the virome associated to fungi isolated from marine organisms assessed on the seagrass Posidonia oceanica and on the marine animal Holothuria poli. We provide detailed protocols for fungal isolation, fungal growth, and total RNA extraction. Ribosomal RNA depletion, cDNA library synthesis and normalization, and sequencing runs on different platforms are part of the protocols that are generally outsourced and therefore are not described in this chapter. We describe, instead, how raw reads are assembled into contigs and how to search for putative viral sequences. Furthermore, we detail qualitative checks to infer the existence of the virus as a replicative biological entity.
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References
Pearson MN, Beever RE, Boine B, Arthur K (2009) Mycoviruses of filamentous fungi and their relevance to plant pathology. Mol Plant Pathol 10:115–128
Marquez LM, Redman RS, Rodriguez RJ, Roossinck MJ (2007) A virus in a fungus in a plant: three-way symbiosis required for thermal tolerance. Science 315:513–515
Nerva L, Varese GC, Falk BW, Turina M (2017) Mycoviruses of an endophytic fungus can replicate in plant cells: evolutionary implications. Sci Rep 7:1908
Panno L, Voyron S, Anastasi A, Varese GC (2010) Marine fungi associated with the sea grass Posidonia oceanica L.: a potential source of novel metabolites and enzymes. J Biotechnol 150:S383–S384
Nerva L, Ciuffo M, Vallino M, Margaria P, Varese GC, Gnavi G, Turina M (2016) Multiple approaches for the detection and characterization of viral and plasmid symbionts from a collection of marine fungi. Virus Res 219:22–38
Marzano SYL, Nelson BD, Ajayi-Oyetunde O, Bradley CA, Hughes TJ, Hartman GL, Domier LL (2016) Identification of diverse mycoviruses through metatranscriptomics characterization of the viromes of five major fungal plant pathogens. J Virol 90:6846–6863
Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388:1–7
Nakayashiki H, Nguyen QB (2008) RNA interference: roles in fungal biology. Curr Opin Microbiol 11:494–502
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Chen Z (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652
Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, MacManes MD (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512
Yutin N, Colson P, Raoult D, Koonin EV (2013) Mimiviridae: clusters of orthologous genes, reconstruction of gene repertoire evolution and proposed expansion of the giant virus family. Virol J 10:106
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120
Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829
Schulz MH, Zerbino DR, Vingron M, Birney E (2012) Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels. Bioinformatics 28:1086–1092
Domsch KH, Gams W, Anderson TH (eds) (1980) Compendium of soil fungi, vol 1. Academic Press, London
Kiffer E, Morelet M (eds) (1997) Les deutéromycètes: Classification et clés d’identification générique. INRA, Paris
Von Arx JA (ed) (1981) The genera of fungi sporulating in pure culture. J. Cramer, Vaduz
Geiser DM, Frisvad JC, Taylor JW (1998) Evolutionary relationships in Aspergillus section Fumigati inferred from partial β-tubulin and hydrophobin DNA sequences. Mycologia 90:831–845
Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–1330
Samson RA, Seifert KA, Kuijpers AFA, Houbraken JAMP, Frisvad JC (2004) Phylogenetic analysis of Penicillium subgenus Penicillium using partial β-tubulin sequences. Stud Mycol 49:175–200
Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553–556
White TJ, Bruns T, Lee SJWT, Taylor JW (eds) (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications, 18. Academic Press, New York, p 315–322
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Nerva, L., Varese, G.C., Turina, M. (2018). Different Approaches to Discover Mycovirus Associated to Marine Organisms. In: Pantaleo, V., Chiumenti, M. (eds) Viral Metagenomics. Methods in Molecular Biology, vol 1746. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7683-6_8
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DOI: https://doi.org/10.1007/978-1-4939-7683-6_8
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